Vol. 12, Issue 10, 1591-1598, October 2002

RESOURCES
An AscI Boundary Library for the Studies of Genetic and Epigenetic Alterations in CpG Islands

¹ Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics, ² Department of Pathology, ³ Department of Veterinary Biosciences, and the Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA; ⁴ Medizinische Universität zu Lübeck, Medizinische Klinik II, Ratzeburger Allee 160 23538 Lübeck, Germany; ⁵ Klinik und Poliklinik für Kinderheilkunde, Pädiatrische Hämatologie/Onkologie, Universitätsklinikum Münster, 48149 Münster, Germany; ⁶ Children's Hospital, Oakland Research Institute, Oakland, California 94609, USA

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.

[Supplemental material is available online at http://www.genome.org.]